The present invention relates generally to the separation of particles suspended in solution based upon unique characteristics of the various particles such as the shape, size and/or deformability, and more particularly to the selective separation or filtration of cells, cell components or fragments thereof which have one or more various unique physical characteristics.
Techniques for the separation of constituents of various medical/biological fluids, such as whole blood, are in wide use for many diagnostic, therapeutic, and other medically-related, applications. For example, centrifugal separation based upon the different densities and settling velocities of the constituent components to be separated is well known. The CS-3000 separator, sold by Baxter Healthcare Corporation of Deerfield, Ill., is one example of a centrifugal separator that has been very successfully used in separating whole blood into constituent components, such as red blood cells (RBC), white blood cells (WBC), platelets, and plasma for collection or depletion of the desired components from a donor or patient. While centrifugation has proven to be a generally satisfactory method of achieving separation, in certain applications the purity of the separated components is not as high as desired due to the very close and/or overlapping densities and settling velocities of the different suspended particles.
Mesh and aggregate structures and membranes are also used to remove particles from suspension. Typically such filters exhibit substantial surface area and/or roughness that can cause particle damage in biological suspensions, e.g., RBC hemolysis and platelet activation in blood.
Separation of biological fluids using a filter membrane with a nominal pore size is also common. For example, it is widely known that a filter membrane having 0.22 micron nominal pore size can be used to filter out assorted bacteria and the like from a liquid. Such membranes, also sometimes called capillary pore membranes, are available in polyester and polycarbonate material from, e.g., Nuclepore Corporation, and in polysulfone from Gelman Sciences, Inc. Such filter membranes have also been used to filter the cellular components of blood (sometimes called the xe2x80x9cformedxe2x80x9d components) from liquid plasma, i.e. xe2x80x9cplasmapheresis.xe2x80x9d
While these membranes have worked satisfactorily in certain applications, such filter membranes have only a nominal pore size, as distinguished from pores of precise and consistent size, shape, and relative spacing to one another. Indeed, it is not uncommon for such nominal pore size membranes to include xe2x80x9cdoubletsxe2x80x9d (i.e., overlapping, non-conforming pores) which would allow passage through the membrane of particles larger than the nominal pore size. To be useful in performing procedures in which particles in a solution are xe2x80x9ccleansedxe2x80x9d of undesirable particles, the undesirable particles being several times larger than the desired particles, filter membranes must exhibit virtually no doublets.
The occurrence of doublets in prior art filter membranes, due to their fabrication techniques, has forced a compromise in their design. Specifically, in order to keep the occurrence of doublets to an acceptable low level, the mean pore-to-pore spacing must be relatively large, which limits the porosity (i.e., the ratio of the total pore area to the total membrane area) of these prior art membranes to about 7% and less. Generally, a lower porosity results in a lower flow rate through the filter membrane. Thus, although a filter membrane having a nominal pore size is suitable for defining an average or nominal maximum particle size that passes through the filter membrane, such membranes are not precisely sized to permit selective filtration of particles of comparable size based on other unique characteristics such as shape or deformability, and have significant drawbacks that limit their application.
A further difficulty with membrane separation of biological and other fluids is impairment of flow through the membrane due to the fouling or clogging of the filter membrane. Such fouling or clogging generally results from the deposition on the surface of the filter membrane of particles too large to pass through the membrane and plugging of the pores. Various methods are known for reducing or preventing the clogging of such membranes. For example, U.S. Pat. No. 5,194,145 to Schoendorfer, herein incorporated by reference, discloses a xe2x80x9ccouette flowxe2x80x9d filter system in which the extraction of filtrate is accomplished through a membrane mounted on a cylindrical rotor within a stationary cylindrical cell. The relative movement between the two concentric cylinders generates a surface velocity that establishes vigorous vortices at the surface of the rotor. These vortices, called Taylor vortices, constantly sweep the membrane surface to limit cell deposition, while continuously replenishing the medium to be filtered.
A different technique to reduce membrane fouling is disclosed in U.S. Pat. No. 4,735,726 to Duggins, herein incorporated by reference. This patent discloses a method and apparatus for carrying out plasmapheresis by conducting blood over the surface of a microporous membrane in reciprocatory pulsatile flow by a peristaltic oscillator or other suitable pump for causing reciprocatory pulsations.
More specifically, Duggins discloses a filter housing having a blood flow region between two plasma flow regions. A central blood inlet port is connected to the blood flow region of the housing, while a blood collection channel is connected to a plasma-depleted blood outlet port, and a plasma collection port is connected to a plasma outlet port. A pair of membranes is disposed between each plasma flow region so that there is a blood flow path between the membranes. Blood is conducted in a forward direction (i.e., away from its source) over the first surface of each filter membrane by, e.g., a rotary peristaltic pump, a piston or syringe pump, or a plunger or hose pump. Blood flow is pulsed in a reciprocatory fashion by a peristaltic oscillator connected to the housing through ports connected to areas near the end of the flow path. As a result, blood can be conducted in the forward direction and in a reverse direction over a first surface of each membrane at a net positive transmembrane pressure, while reducing the transmembrane pressure during the forward and reverse conduction of the blood. The frequency and volume of the reciprocatory pulses are selected to maximize the flow of plasma through the membranes without causing extensive blood trauma. The plasma which passes through each membrane is collected, while the plasma depleted blood is recirculated to the blood flow region.
More recently, it has been possible to make microporous filter membranes with pores having precise size and shape through techniques such as those shown in U.S. application Ser. No. 08/320,199, entitled xe2x80x9cPorous Microfabricated Polymer Membrane Structurexe2x80x9d, filed Oct. 7, 1994, now U.S. Pat. No. 5,807,406, having the same assignee as the present invention and which is incorporated herein by reference. The aforesaid application generally discloses a process for microfabricating precise membranes using etchable polyimide film on a silicon substrate. A polymer film layer is made from a photoimageable polyimide material. The film is processed using negative photoresist techniques or etchable membrane fabrication technique to create a predefined geometric pattern of holes and intermediate spaces defining strands.
Alternatively, other processes, such as positive photoresist techniques, RIE (Reactive Ion Etching), LIGA (an abbreviation of the German for lithographic, galvanoformung, abformung, or in English, lithography, electroforming, and molding), may be used to create filter membranes with extremely small pore size (e.g., less than 10 microns) and having virtually zero doublets that are exceptionally uniform, with a high degree of consistency from one pore to the next. Further, electron beam and ion etch techniques also are possible means to produce precision, high porosity membranes with exceptionally small pores. With the doublet problem essentially eliminated by these various fabrication techniques, organic membrane structures with very high porosities (exceeding 35% and potentially achieving 80%) can be generated with the pore area limited only by structural considerations.
It is a general object of the invention to provide an improved method and apparatus utilizing precise pore size and shape membranes for selectively separating the particles or components in a medical, biological or other suspension based on the size, shape, deformation characteristics, or other unique characteristic of the various components to be separated.
More specifically, it is an object of the present invention to provide an improved method and apparatus for selectively separating the various components of whole blood, such as red blood cells, white blood cells, and platelets, or substances that may be found in whole blood, based upon their size, shape, and/or deformation characteristics.
It is a further object of the invention to provide method and apparatus employing precise pore size which include means for preventing fouling or clogging of the surface of the filter membrane.
These objects, as well as others which will become apparent upon reference to the following detailed description and accompanying drawings. Before turning to the detailed description, and for purposes of summary only, one aspect of the present invention is embodied in a method for separating a suspension comprising at least two types of particles, which are differently sized or shaped. Such particles may be biological cells or cellular components and, more particularly, animal cells or cellular components characterized by having a non-rigid cell membrane, free of a rigid outer cell wall, and consequently being subject to trauma when stressed. The first type of particle may also be, but is not necessarily, deformable at a relatively lower force and/or faster rate than the second type of particle. This method comprises providing a filter membrane having substantially precisely dimensioned pore sizes, with the pores being dimensioned to allow passage of the first type of suspended particle without distortion or with only minimal distortion and passage of the second type of particle only with substantial distortion. Because the filter membrane has precisely dimensioned pores, with the spacing between the pores being maintained despite the smaller interval between the pores, the porosity of the membrane may be much greater than nominal pore size membranes, with less internal pathway variability. This allows faster filtration rates and/or smaller membranes for a given filtration rate, reducing the exposure time of cells within the shear environment of the separator and, consequently, reducing particle damage (such as WBC damage, platelet activation, and/or RBC hemolysis). Similarly, the smooth membrane surface and the smoothness of the internal pathways of the pores permit more consistent fluid shear near the membrane surface and further reduces the exposure time of the particles to the pores.
In this method, the membrane is contacted with the suspension to allow the passage of the first type of particle and to block passage of the second type of particle. To enhance the passage of the first type of particle through the pores, in one class of embodiments the membrane thickness may be small relative to the first type of particle. In another class of embodiments the membrane thickness may be large relative to the second type of particle in order to further inhibit deformation of the second type of particle. To enhance passage of the first type of particle and blockage of the second type of particle, the time that the suspension and membrane are in contact, the force of contact between the suspension and membrane and/or the relative movement between the suspension and membrane may be selectively varied, either alone or in combination.
In accordance with another aspect of the present invention, a method may be provided for filtering a suspension comprising at least two types of differently sized and/or shaped particles that differ in deformability characteristics. In this method, the filter membrane has substantially precisely dimensioned pores and may also have a very high porosity. The suspension and the precise pore size filter membrane are brought into contact with each other with a force or for a time sufficient to allow deformation of the first type of particle for passage through the pores, but insufficient to allow deformation of the second type of particle for passage through the pores.
Although the two aspects or methods above are referred to separately, they are not necessarily separate and may be employed in combination. For example, it is within the scope of the present invention to employ a precise pore size membrane that has precise size, and wherein the solution to be filtered includes first and second types of particles of different shape and different deformation characteristics. The precisely dimensioned pores may be of a shape conforming generally to the shape of the first particle only and of a size that requires some deformation of the first particle to pass therethrough. The suspension is brought into contact with the membrane for sufficient time and/or pressure to allow the first particle to deform and pass through the pores, but not the second particle. As in the first-described method, the time that the suspension and membrane are in contact, the force of contact between the suspension and membrane and/or the relative movement between the suspension and membrane may be selectively varied, either alone or in combination, to enhance passage of the first type of particle through the membrane.
In the methods referred to above, the lack of non-conforming pores improves the purity of separation and the very high available porosity improves the process by reducing the suspended particles"" exposure time in the filtration shear field, as the particles pass through the membrane, by a factor of about 3 to 11, thus reducing the trauma to the separated particles due to filtering. The required membrane area is reduced by a similar factor, thus potentially reducing device size and cost substantially, and reducing particle stress related to the time of exposure of the particle to the shear field.
In the methods described above, an additional step of cleaning the upstream surface of the membrane may be included to prevent accumulation of the second type of particle on the surface of the membrane, which may result in clogging or blocking of the pores. By way of example, and not limitation, the cleaning step may be performed by flowing suspension across, i.e., parallel to, the surface of the filter membrane, creating turbulence on the surface of the membrane to sweep the clogging particles off the surface, or relatively oscillating the membrane and suspension to flush the second type of particles off the surface of the membrane.
The present invention is also embodied in apparatus for carrying out the above methods. An apparatus for performing the first mentioned method, for example, may comprise a filter membrane having substantially precisely dimensioned pores (which includes membrane thickness requirements), shaped to correspond substantially to the shape of the first type of particle and to allow passage thereof without or upon only minor deformation of the first type of particle, but to block passage of the second type of particle. Means is provided for bringing the suspension and the membrane into contact sufficient to permit passage of the first type of particle through the membrane, but insufficient to allow for substantially any of the second type of particle to pass through the membrane pores. The aforesaid means may also reduce clogging of the membrane pores.
Apparatus for performing the second identified method may also comprise a filter membrane having substantially precisely dimensioned pores (which includes membrane thickness requirements). In the second embodiment, the membrane pores are precisely dimensioned to allow passage of the first type of particle only upon deformation and the first type of particle is deformable at a faster rate than the second type of particle. Like the apparatus for performing the first method, the second apparatus also includes means for bringing the suspension into contact with the membrane with a forcexe2x80x94either direct or shearxe2x80x94and for a time sufficient to permit the first type of particle to deform and pass through the membrane, but insufficient to allow deformation of substantially any of the second type of particle for passage through or plugging of the membrane pores. Of course, great care is necessary in the selection of membrane materials for separation, concentration, or removal of particles from suspension, particularly in the case of biological suspensions such as blood. Hydrophilic materials, such as polycarbonate, or special surface coatings or modifications, in addition to anticoagulants, are typically required for blood products to avoid or minimize platelet activation, blood cell aggregation, clotting, and/or hemolysis.
As set forth more fully below, these methods and apparatus find particular application in the selective separation of the formed elements of blood (red cells, white cells and platelets) from one another or the separation of formed components from the plasma, the liquid in which they are suspended. If, for example, in the first method and apparatus the liquid to be separated is whole blood, and it is specifically desired to separate the white blood cells from the red blood cells, a filter membrane may be provided that has pores precisely rectangularly dimensioned to measure approximately 1.8 microns to 3.5 microns by approximately 6.0 microns to 14.0 microns to allow passage of red blood cells without deformation or with only minor deformation and white blood cells only upon substantial deformation of the white blood cells. It is known that white blood cells deform at a substantially slower rate than red blood cells when subjected to the same force. The whole blood and filter membrane are brought into contact for a time sufficient, or with a force sufficient, or a combination of time and force sufficient, to allow any required deformation of the red blood cells for passage through the pores in the filter membrane, but insufficient to deform substantially all of the white blood cells for passage through the pores.
The description above is intended only by way of summary. A more detailed description of the various features and advantages of the present invention are set forth below.